As imaging technology improves via advances including better optics, higher resolutions, and the ability to image deeper into tissue with three dimensional results, there is a higher demand for more advanced imaging protocols to image tissue on a large scale.
One of the biggest issues with imaging the entirety of an organ, or even a relatively small sample of tissue (e.g., 1 mm×1 mm), is the difficulty of using traditional immunohistochemical (IHC) techniques to achieve the large scale results that are desirable. Typical IHC procedures require the embedding of the sample into paraffin or snap frozen into an optimum cutting temperature (OCT)-based block. Paraffin is useful for subsequent antibody labeling but will remove any endogenous fluorescence localized in the tissue. The frozen blocks will retain a certain amount of fluorescence, however they will damage or destroy a lot of microstructures in the tissue. Once embedded with one of the above methods, the blocks then have to be sliced into extremely thin, approximately 4 micrometers (μm), slices that are processed and put onto slides for staining and imaging. With 4 μm slices, it would take 250 separate slices for a 1 millimeter (mm) thick tissue, which would have to be put onto slides for subsequent staining and imaging. This procedure would therefore take an unacceptable amount of time for the tissue preparation alone. Time is not the only concern with these methods, however. By cutting the tissue, damage to the edges of each tissue slice is inevitable. This damage will create “gaps” and damaged areas in between each tissue slice when all of the slices are aligned and reconstructed. Furthermore, the actual imaging of these 250 tissue slices would take an unacceptable amount of time. Most oil-dipping objectives don't even have a working distance long enough to image through the entirety of a 4 μm thick tissue slice. Even if all of the slices are fully imaged through the entirety of the slice thickness, the ability to align and accurately reconstruct the sample would be extremely difficult.
Due to the aforementioned difficulties that arise when performing tissue analysis on a larger (macro) scale, researchers have turned to using multiphoton imaging with a whole mounted, unsectioned specimen for deep tissue imaging. This technique allows one to use a planar focused laser to optically slice through the tissue, obviating the need for sectioning. Such a technique, however, carries with it a vast number of limitations and problems that arise when performing deep tissue imaging. One of the most notable issues is the inability to limitlessly image into the depth of the tissue. Under the most optimal conditions, imaging of tissue up to 1000 μm in very low autofluorescent tissue has been achieved [Levene, et al. J. Biomed Opt, 2010, 15(3):036017]. However, attaining such a result is extremely difficult and rare with all tissues, most notably the brain. Realistically, most deep tissue imaging can currently be performed up to a range of about 300-500 μm.
The inability to image deeper into the tissue arises from light absorption, and light scattering caused by lipids in the tissue. Not only do the lipids create an extreme autofluorescent signal, specifically in adipose or in tissues with high lipid content, such as liver, but they also create light scattering of the fluorescent signal and the input laser signal. There have been many attempts at combating the light scattering issue that arises from lipids [Nonlinear optical microscopy: use of second harmonic generation and two-photon microscopy for automated quantitative liver fibrosis studies. Sun W, Chang S, Tai D C, Tan N, Xiao G, Tang H, Yu H., J Biomed Opt. 2008 November-December; 13(6):064010].
One of the more basic attempts at clearing tissue has been through the use of sucrose solutions to clear the tissue [SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Ke M T, Fujimoto S, Imai T. Nat Neurosci. 2013 August; 16(8):1154-61. Correlation between collagen solubility and skin optical clearing using sugars. Hirshburg J, Choi B, Nelson J S, Yeh A T. Lasers Surg Med. 2007 February; 39(2):140-4]. While there has been some success with very small pieces of tissue, large samples cannot be used. There are multiple concerns involved with this clearing technique. Sucrose causes a dehydration of the tissue, ultimately causing shrinkage of the actual tissue, most likely due to extreme osmolality changes. The shrinkage of the tissue does not provide accurate information about the structure of the tissue, specifically the microstructure. This technique does not permit the ability to label the tissue with antibodies. Also, the sucrose solutions do not provide a good medium for maintaining endogenous fluorescence. The light transmittance with sucrose-cleared tissues is also extremely poor. True and complete clearing using sucrose clearing is unattainable.
Glycerol clearing has also been performed on smaller sample sizes [A rapid approach to high-resolution fluorescence imaging in semi-thick brain slices. Selever J, Kong J Q, Arenkiel B R., J Vis Exp. 2011 Jul. 26; (53)]. The limitations of this technique are very similar to those mentioned in the sucrose technique. Glycerol can only be used on extremely small and thin samples. There is a smaller volume change in the sample when using glycerol; however, volume change does still occur. Antibody labeling cannot be utilized with this technique. Glycerol is also a very poor medium for endogenous fluorescence since it has been shown to quench a majority of the fluorescence. The light transmittance with glycerol is actually much better than sucrose when using a proper objective that matches the glycerol refractive index (RI) in the tissue. True complete clearing using glycerol clearing is typically unachievable.
Another technique that has shown relative success in clearing tissue is called benzyl alcohol and benzyl benzoate (BABB). The technique also uses tetrahydrofuran (THF) to aid in the clearing process. While this technique does in fact actually “clear” the tissue, there are many concerns. BABB causes the most drastic tissue shrinkage out of all the clearing techniques demonstrated in literature, thus creating the worst tissue structure representation. BABB also entirely quenches the endogenous fluorescence of the sample. The THF and BABB solutions are also highly caustic to use, thus extreme care must be utilized to perform this technique. The light transmittance through BABB cleared tissue is still rather poor although better than sucrose or glycerol. Antibody labeling is also unachievable with this technique.
A more recent technique that introduced an era that is expanding our abilities for deep tissue imaging is called SCALE [Hama et al., Nature Neuroscience 14(11): 1481-1490 (2011)]. SCALE is a clearing reagent containing a concentrated urea solution in which the sample incubates until the tissue is cleared. Such incubation can require weeks to months with regular media exchanges. One mechanism by which this may occur is through the superhydrating effects that the solution has on the tissue. The SCALE technique has allowed researches to image up to 8 mm through brain tissue with a resolution better than what is achieved when deep tissue imaging in a normal whole mount tissue. Fluorescence is adequately maintained and the technique is easy to perform. However, the major limitations are (i) the inability to do antibody labeling, (ii) the approximate 1.5× volume expansion of the tissue, and (iii) the amount of time that it takes to perform the technique. Clearing a whole mouse brain can take anywhere from 4 weeks to 6 months. This technique also led to the creation of a SCALE-specific objective series from Olympus that consists of 4 mm and 8 mm working distance objectives that are specifically refractive index matched to the SCALE solution. While this technique has provided some advantages in the field of tissue clearing, it also possesses limitations. For example, SCALE results in a denaturation of the majority of proteins from the sample, there is an inability to probe with antibodies in the SCALE solution, and the majority of fluorescent signal is lost in a time dependent manner.
A recent clearing technique that has been demonstrated is SeeDB. This technique has been shown to be successful at clearing tissue with a technically simple method. SeeDB utilizes gradient washes of the tissue in Fructose/1-Thioglycerol solutions for rather short periods of time, approximately 12 hours. This creates a cleared specimen in about 7 days. SeeDB is also able to retain fluorescent signal in the tissue. SeeDB also provides the best light transmittance compared to earlier techniques, as well as a lack of shrinking or expansion of the actual tissue. SeeDB does not, however, allow antibody staining and the cleared sample can only be maintained in the final clearing solution for a maximum of 7 days, at which time the sample has to be washed free of fructose. There have also been a lot of problems reported with autofluorescence and browning developing in the tissue due to a maillard reaction. This technique has also not been proven to completely clear the entirety of an organ; in fact, the technique performs best with tissue slices that are about 1-2 millimeters in thickness. One of the main benefits of this technique is the high refractive index that is achieved with the final sample, 1.51. This matches most oil immersion objectives and allows for an incredibly high optical resolution. A custom high refractive index objective has also been created by Olympus to accommodate this higher RI.
Probably the most notable of clearing techniques to recently be published is called CLARITY. CLARITY takes a completely different approach to clearing the tissue than those mentioned above. CLARITY actually delipidates the tissue, thus removing the cause of the light scattering: lipids. In order to achieve such delipidation with minimal loss of proteins, the sample is embedded into an acrylamide-based gel that is polymerized into the sample. This causes a crosslinking effect that binds the proteins and such, without binding to the actual lipids. Such crosslinking allows for removal of the lipids with limited damage to the rest of the components of the tissue. It has been shown that the microstructures of the tissues are kept intact. CLARITY utilizes an Electrophoresis Tissue Clearing Chamber (ETC), with an SDS-based buffer, that accelerates the removal of the lipids from the sample. The cleared tissue is then washed and the refractive index matched in either FocusClear™ or 80% glycerol for imaging. The major benefit of CLARITY is that antibody penetration is possible due to the complete removal of the lipids from the sample. It has been demonstrated that antibodies can be probed, imaged, and stripped for subsequent antibody labeling experiments. The ability to keep the tissue structurally accurate is also another benefit. The problems with CLARITY arise with the variety of tissues that are compatible with the technique. CLARITY was developed primarily to focus on clearing the mouse brain for brain mapping experiments. While it has been shown that this works for nervous tissue, it is not as compatible with other organs/tissues in the body. Attempts at using the CLARITY technique on other parts of the mouse were met with very little success. (See, e.g., the CLARITY Resources website.) Other tissues burn, form black on the outside, turn brown/yellow, and even degrade. Researchers have also reported difficulty even when attempting to clear the brain (see, e.g., the CLARITY Resources website). The protocol itself is technically difficult to perform, while also being tremendously costly. There is also an inability to perform the clearing technique on multiple samples if only one chamber is created. It is therefore an ineffective technique if one is attempting a round of experiments that would require clearing of a dozen or more samples. Another confounding factor is the mounting medium. FocusClear™ has been shown to be a great product for tissue clearing; however, it is prohibitively costly. FocusClear™ has also been demonstrated to reduce fluorescent signals, quenching signals such as green/red fluorescent protein (GFP/RFP). (See, e.g., the CLARITY Resources website). An alternative to FocusClear™ is glycerol, which, as stated above, is not very compatible with fluorescence. Researchers in general have also been reporting a significant loss of endogenous GFP fluorescence when performing CLARITY. (See, e.g., the CLARITY Resources website.)